TY - JOUR
T1 - Transcriptome analysis approaches for the isolation of trichome-specific genes from the medicinal plant Cistus creticus subsp. creticus
AU - Falara, Vasiliki
AU - Fotopoulos, Vasileios
AU - Margaritis, Thanasis
AU - Anastasaki, Thalia
AU - Pateraki, Irene
AU - Bosabalidis, Artemios M.
AU - Kafetzopoulos, Dimitris
AU - Demetzos, Costas
AU - Pichersky, Eran
AU - Kanellis, Angelos K.
N1 - Funding Information:
Acknowledgments We thank G. Papagiannakis for participating in the construction of Cistus microarrays; we are grateful to Dr. C. Sallaud (Librophyt, France) for providing the hexane extract of Solanum hirsutum and to Dr. M. Lazari for her advice during the Cistus hexane extractions. We acknowledge the help of E. Argyro-poulou in isolating trichomes shown in Fig. 1b–e. This work was funded by GR-USA bilateral grant and the 99Ε∆637 and 01Ε∆416 research projects, implemented within the framework of the ‘‘Reinforcement Programme of Human Research Manpower’’ (PENED) and co-financed by National and Community Funds (25% from the Greek Ministry of Development-General Secretariat of Research and Technology and 75% from E.U.-European Social Fund). This work was also partially supported by grants to AKMON (AP.6260 EFA1250/17-5-2004) co-funded by 70% from the European Union Regional Development Fund and by 30% from the Operational Programme Competitiveness of the Greek Ministry of Development.
PY - 2008/12
Y1 - 2008/12
N2 - Cistus creticus subsp. creticus is a plant of intrinsic scientific interest due to the distinctive pharmaceutical properties of its resin. Labdane-type diterpenes, the main constituents of the resin, exhibit considerable antibacterial and cytotoxic activities. In this study chemical analysis of isolated trichomes from different developmental stages revealed that young leaves of 1-2 cm length displayed the highest content of labdane-type diterpenes (80 mg/g fresh weight) whereas trichomes from older leaves (2-3 or 3-4 cm) exhibited gradual decreased concentrations. A cDNA library was constructed enriched in transcripts from trichomes isolated from young leaves, which are characterized by high levels of labdane-type diterpenes. Functional annotation of 2,022 expressed sequence tags (ESTs) from the trichome cDNA library based on homology to A. thaliana genes suggested that 8% of the putative identified sequences were secondary metabolism-related and involved primarily in flavonoid and terpenoid biosynthesis. A significant proportion of the ESTs (38%) displayed no significant similarity to any other DNA deposited in databases, indicating a yet unknown function. Custom DNA microarrays constructed with 1,248 individual clones from the cDNA library facilitated transcriptome comparisons between trichomes and trichome-free tissues. In addition, gene expression studies in various Cistus tissues and organs for one of the genes highlighted as the most differentially expressed by the microarray experiments revealed a putative sesquiterpene synthase with a trichome-specific expression pattern. Full length cDNA isolation and heterologous expression in E. coli followed by biochemical analysis, led to the characterization of the produced protein as germacrene B synthase.
AB - Cistus creticus subsp. creticus is a plant of intrinsic scientific interest due to the distinctive pharmaceutical properties of its resin. Labdane-type diterpenes, the main constituents of the resin, exhibit considerable antibacterial and cytotoxic activities. In this study chemical analysis of isolated trichomes from different developmental stages revealed that young leaves of 1-2 cm length displayed the highest content of labdane-type diterpenes (80 mg/g fresh weight) whereas trichomes from older leaves (2-3 or 3-4 cm) exhibited gradual decreased concentrations. A cDNA library was constructed enriched in transcripts from trichomes isolated from young leaves, which are characterized by high levels of labdane-type diterpenes. Functional annotation of 2,022 expressed sequence tags (ESTs) from the trichome cDNA library based on homology to A. thaliana genes suggested that 8% of the putative identified sequences were secondary metabolism-related and involved primarily in flavonoid and terpenoid biosynthesis. A significant proportion of the ESTs (38%) displayed no significant similarity to any other DNA deposited in databases, indicating a yet unknown function. Custom DNA microarrays constructed with 1,248 individual clones from the cDNA library facilitated transcriptome comparisons between trichomes and trichome-free tissues. In addition, gene expression studies in various Cistus tissues and organs for one of the genes highlighted as the most differentially expressed by the microarray experiments revealed a putative sesquiterpene synthase with a trichome-specific expression pattern. Full length cDNA isolation and heterologous expression in E. coli followed by biochemical analysis, led to the characterization of the produced protein as germacrene B synthase.
KW - Cistus creticus subsp. creticus
KW - DNA microarrays
KW - EST analysis
KW - Gene expression
KW - Germacrene B synthase
KW - Trichomes
UR - http://www.scopus.com/inward/record.url?scp=54849404456&partnerID=8YFLogxK
U2 - 10.1007/s11103-008-9399-0
DO - 10.1007/s11103-008-9399-0
M3 - Article
C2 - 18819010
AN - SCOPUS:54849404456
SN - 0167-4412
VL - 68
SP - 633
EP - 651
JO - Plant Molecular Biology
JF - Plant Molecular Biology
IS - 6
ER -